Figure 4.

Arm interacts with CBP in vitro and in vivo. (A) Western blot with anti-CBP antisera demonstrating that GST-Arm, but not GST alone, is able to pull down fly CBP from extracts of 293 cells expressing CBP. (B) Arm and fly CBP interact when overexpressed in Kc cells. Cells were transfected with plasmids expressing V5-tagged Arm^* and Flag-tagged CBP. Forty-eight hours post-transfection, cell extracts were prepared and immunoprecipitated with V5, Flag and control IgG antibodies and the precipitates were analyzed by Western blot with anti-V5 antibody. Flag precipitation pulled down a significant portion of Arm^*-V5, compared with control IgG. (C) Endogenous Arm and CBP interact. Kc cells were treated with WCM or control media for 4 h before extract preparation. Proteins were immunoprecipitated with anti-CBP antisera or control IgG and Western blot were analyzed with anti-Arm antibody. CBP interacts with endogenous Arm in Wg-stimulated cells. (D) Arm and CBP interact directly in vitro. Bacterially expressed GST or GST–CBP fragments of the indicated residues were incubated with ³⁵S-Met-labeled Arm C-terminal fragment (residues 429–815). Precipitated proteins were analyzed together with 5% of the input material by SDS–PAGE and autoradiography. Three CBP fragments bound to the C-terminal fragment of Arm, while fragments comprising residues 1200–2550 show similar binding as the GST-negative control. All experiments shown were performed multiple times, with similar results obtained.